Cell cultures are used widely in science and industry for purposes varying from protein production, food and beverage fermentation to pharmaceutical production. Cell cultures are often used in laboratory settings for research and disease diagnosis and, with the advent of genetic engineering, modified organisms are commonly used for the production of medical proteins, antibiotics, cytokines, insulin, hormones, and other biomedically-important molecules.
Fermentation is one of the most important processes in the pharmaceutical industry. Industrial fermentation loosely refers to the breakdown of organic substances and re-assembly into other substances. Fermenter culture in industrial capacity often refers to highly oxygenated and aerobic growth conditions. For instance, penicillin, which brought enormous profits and public expectations, was produced industrially using a deep fermentation process. When a particular organism is introduced into a selected growth medium, the medium is inoculated with the particular organism. Growth of the inoculums does not occur immediately, but follows a lag phase. Subsequently the rate of growth of the organism steadily increases, for a certain period known as the log or exponential phase. The rate of growth eventually slows down due to the continuously falling concentrations of nutrients and the continuously increasing (accumulating) concentrations of toxic substances. This phase, where the increase of the rate of growth is checked, is the deceleration phase. After the deceleration phase, growth ceases and the culture enters a stationary phase or steady state.
The fermentation industry relies on bacterial and fungal cultures to produce alcoholic beverages, process a wide variety of foods, neutralize toxic spills, break down liquid and solid wastes, create biologically-significant compounds, and transform corn and other raw materials into bio-fuels such as bio-ethanol. Bio-ethanol is a biodegradable, non-toxic renewable energy source which is expected to contribute significantly to solving the world's energy crisis (Soetaert and Vandamme, 2009). Bio-ethanol is produced mainly from agricultural products and food wastes containing sugar, starch, or cellulose which can be fermented and distilled into ethanol. However, the conversion of sugar or starch to bio-ethanol requires large areas of land to grow raw materials, and may negatively impact the food industry by removing available food (Pandey, 2008). Lignocellulosic ethanol can be produced from a variety of unutilized feedstock, with typical sources of lignocellulosic biomass including bugasse of sugar cane, corn stover, grasses, woody biomass, industrial wastes, and dedicated woody crops (Pandey, 2008). Due to the high crystallinity of lignocelluloses, it is unfortunately difficult to hydrolyze into individual glucose subunits for fermentation and the cost of cellulase enzymes required for hydrolysis can account for 30-50% of the total operation costs (Chen and Qiu, 2010; Yang et al., 2010).
In almost all useful applications of cell cultures, the rate at which the desired product is produced is limited only by the rate at which protein expression occurs, and the growth rate of the cells used in production. Industrial fermentation most often takes place in a specially designed environment in which cells are grown in suspension. The fermenter maximizes cell growth by carefully maintaining optimal temperature and agitating the contained mixture to ensure transfer of nutrients into and metabolic byproducts out of the cell. However, it is difficult to ensure that the turbulence in the tank is pervasive enough to affect the cells on the microscopic level. There is often a relatively stagnant region directly adjacent to the walls of the cells and the fermenter itself. This naturally has a negative effect on the nutrient and toxin transfer to and from the cells, and reduces the rate of protein expression, lowering the overall productivity of the fermentation process.
As fermentation is a widely practiced art and has many industrial applications, it is clear that some method of improving the rate of cell protein expression, ethanol, and biodrugs production would be desirable.
Ultrasound is broadly defined as sound waves at a frequency above the normal hearing range, or a frequency greater than 20 kHz (Khanal et al., 2007). Ultrasound is traditionally used in medical diagnosis, such as fetal imaging, which employs frequencies between 2 MHz and 18 MHz and therapeutic treatment of injured muscles, ligaments and tendons, using frequencies between 1 to 5 MHz.
Ultrasonic stimulation creates “microcavitation” or the creation of minute bubbles in a liquid known as “microcavities.” With each sound wave, these bubbles expand and contract, creating tremendous force and turbulence on a microscopic scale. In some cases, this sound wave is powerful enough to collapse the cavities, which causes even more extreme turbulence, high temperatures, and free radicals in the vicinity of the former cavity. These collapses are powerful enough to dislodge or even destroy cells.
Ultrasonic applications rely on these processes. One common use of ultrasound is as an effective cleaning agent. If the intensity is high enough, collapse cavitation is the dominant factor in the cells' environment. This can strip or even kill harmful bacteria from a surface. The effectiveness of this technique has been proven by applying ultrasound to one end of a glass tube using frequencies around 100 kHz and intensities around 40 W/cm2. It was found that approximately 88% of the bacteria were removed from the surface of the tube. Similar experiments have been carried out in a variety of situations, including stripping biofilms from reverse osmosis membranes. Ultrasound is now actively sold to laboratories as a cleaning aid.
As well as dislodging bacteria, very high intensity ultrasound (>10 W/cm2) has been used to kill suspended bacteria. This relies on collapse cavitation to rend the bacterial membrane.
Applications also exist for low intensity pulsed ultrasound (LIPUS) which generally utilizes an intensity of about 0.1-0.2 W/cm2. LIPUS has been used for repair of bone fractures (Rubin et al., 2001), cell stimulation and differentiation (Yoon et al., 2009), stimulation of growth factors (Kobayashi et al., 2009), protein and fibroblast growth (Doan et al., 1999; Min et al., 2006; Sun et al., 2001; Wood et al., 1997; Zhou et al., 2004), dental tissue formation (Ang et al., 2010; Leung et al., 2004), stem cell proliferation (Gul et al., 2010), sonoporation including ultrasound-mediated gene delivery (Osawa et al., 2009), and biomass pre-treatment before saccharification (Svetlana et al., 2010). It is believed that ultrasonic waves can improve the rate of bone growth and indeed, almost 80% of North American physiotherapists possess ultrasonic emitters for the purpose of encouraging speedy recovery. However, only LIPUS is effective in this situation, with LIPUS devices being currently being marketed for this purpose (see for example, U.S. Pat. No. 4,530,360 to Duarte).
Use of low-intensity pulsed ultrasound to aid the healing of flesh wounds is described, for example, in U.S. Patent Application Publication No. 2006/0106424 A1 to Bachem. The method utilizes ultrasound to increase the phagocytotic action of the human body's macrophages. However, the method provides no solution for the use of ultrasound outside the confines of a wound.
U.S. Patent Application Publication No. 2003/0153077 A1 to Pitt et al. describes a method in which low-intensity ultrasound can stimulate the growth of biofilms and other cells. By balancing the beneficial turbulence produced by collapse cavitation with its accompanying negative effects, it was found that low-intensity ultrasound can improve growth rates of cells by up to 50%. The experimenters tested their findings on human and bacterial cells, using frequencies from about 20 kHz to about 1 MHz and intensities encompassing the range from 1 to 5000 mW/cm2. Unfortunately, though increased cell growth is beneficial to the fermentation process, the parameters investigated by this group do not provide the optimal rate of protein expression in fermentation processes.